The ability of vascular endothelial cells to adjust their cytoplasmic properties under the stimulus of fluid shear stress is a key factor in the physiological control of the blood-vessel wall interface. Adjustments to fluid stress control exchange and cell traffic across the endothelium, influence humoral regulation, and are one of the main determinants of lesion formation in large arteries. Cytoplasmic actin plays a key role in these processes because it serves to define the cell's shape, mechanical properties, and motility. Actin polymer (F-actin) stability, arrangement, and extent of crosslinking and attachment to the substrate and the lumenal membrane determine the local stiffness of the cytoplasm. There exists today no measurements of the dynamics of actin in endothelial cells or the consequence of these dynamics for the mechanics and biology of endothelial cells under flow conditions commensurate with those experienced in vivo. Aim 1 will determine actin dynamics in individual human umbilical vein endothelial cells (HUVEC) before and after the application of steady fluid shear to their surfaces. Cells are microinjected with caged resorufin-actin monomer (G-actin). After incorporation of the caged-actin into the cytoskeleton, spatial uncaging of its fluorophore will yield the F-/G-actin ratio, F-actin turnover time, and the G-actin diffusion coefficient. In Aim 2, F-actin turnover data will be combined with a detailed 3D picture of F-actin structure in endothelial cells determined using high-resolution electron microscopy with the goal of developing a model to explain the mechanical properties of the cell and how these properties are altered by fluid flow. In Aim 3, the measurements of actin dynamics and internal structure will be extended to disturbed and unsteady flow to create a complete biomechanical picture of normal in vivo conditions. These studies will provide new insight into the basic physiology of endothelial structure and function under flow conditions characteristic of those experienced in vivo. They will provide new data on cell stasis and motility as well as the coupling between arterial flow and the cell, and thereby contribute to our understanding of the pathologies associated with atherosclerosis and intimal hyperplasia in vascular grafts.